Normal adult hemoglobin (HbA) is comprised of four polypeptide subunits, two .alpha.-globin chains and two .beta.-globin chains. The .alpha. chains are encoded by a gene on chromosome 16, and the .beta. chains are encoded by a gene on chromosome 11. During fetal life, fetal hemoglobin (HbF) is composed of four polypeptide subunits, similar to HbA. However, instead of two .beta.-subunits, HbF contains two .gamma.-subunits together with two .alpha.-subunits. Gamma-globin is also encoded on chromosome 11, upstream of .beta.-globin.
The expression of the globin genes is regulated during ontogeny. The production of globin begins to switch from HbF, containing .alpha.2.gamma.72, to HbA, containing .alpha.2.beta.2, just before birth. The switching process to HbA is complete usually by 4 months after birth. However, some HbF continues to be produced in normal adults comprising about 1% of the total hemoglobin. As such, any structural or functional defects of the .beta.-globin gene become clinically evident only on completion of the perinatal .gamma.- to .beta.-globin switch.
The .beta.-hemoglobinopathies (globin disorders), such as sickle cell anemia and .beta.-thalassemia (or Cooley's anemia), are among the most common of the genetic disorders, afflicting millions of people world-wide. They are incurable hereditary disorders of hemoglobin structure and synthesis, respectively, that have their origins in mutations affecting the .beta.-globin gene locus or a region controlling its expression. In sickle cell anemia, a point mutation in the DNA sequence of the .beta.-gene involving a replacement of glutamic acid with valine results in a defective .beta.-globin protein which complexes with another of its kind forming polymerized proteins called hemoglobin S (HbS). The tendency towards sickling is dependent on both the relative quantity of HbS in erythrocytes and the level of oxygen tension in the micro environment of the body. These polymerized .beta.-globins cause severe distortions in red cell morphology, membrane damage by blocking capillaries and lead to hemolysis and extreme, although episodic, pain. Other problems include liver involvement such as jaundice, hyperbilirubinemia, and severe anemia. No effective conventional therapies for sickle cell anemia exist and treatment of this disorder is confined to the management of acute pain and the consequences of end-organ damage.
.beta.-thalassemia is characterized by inadequate or absent production of the .beta.-globin chain which is the result of mutations in the gene or in the gene promotor. This results in an accumulation of excess .alpha.-globin chains, which are toxic to red cells and promote cell lysis. This premature destruction of red cells produces a severe anemia. To compensate for this, erythropoiesis expands dramatically resulting in hyperplastic marrow and consequently grossly enlarged bones. These patients require transfusions to sustain life, but the onset of iron overload results in death in most by the third decade of life. As yet, there is no effective conventional therapy for this disorder.
Several lines of evidence suggest that increasing cellular levels of HbF should prove clinically beneficial to sufferers of sickle cell anemia and .beta.-thalassemia. A subpopulation of people with sickle cell anemia has been observed wherein these patients had unusually high levels (&gt;10 to 100%) of HbF and displayed no clinical symptoms of the disorder. Further studies show that patients with slightly increased levels of HbF (above the normal 1%) have more mild or benign clinical symptoms. A level of 4-15% HbF approaches that considered necessary to ameliorate significantly the severity of sickle cell disease. Fetal hemoglobin has been found to decrease or inhibit polymerization of .beta.-globin thus alleviating sickling of the red cells. Any increase in the production of non-.alpha.-globin in the case of .beta.-thalassemia will result in more effective erythropoiesis thus ameliorating this disorder.
To date, several pharmacologic agents have shown a capacity for increasing the HbF synthesis in subjects. These include cell-cycle specific cytotoxic compounds, nucleoside analogues, hematopoietic growth factors and butyric acid derivatives. Cell-cycle specific compounds, such as 5-azacytidine, have resulted in increased levels of HbF. (Charache, S. et al. PNAS; 80:4842-4846, 1983). However these agents are carcinogenic and hence are unattractive as lifelong therapies, particularly for younger patients. Another drug that has been shown to increase HbF is the anti-cancer drug, hydroxyurea; however it also is toxic and not well tolerated by many patients. (Charache, S. et al., Blood; 69: 109-116, 1987.)
Perrine U.S. Pat. No. 4,822,821 (issued Apr. 18, 1989) provides a method for inhibiting the .gamma.- to .beta.-globin switching in fetal or infant subjects afflicted with .beta.-globin disorders by administering .alpha.-amino-n-butyric acid or butyric acid and isomers thereof to the subject prior to the natural completion of the switching process. This patent does not describe the method of increasing HbF using compounds of the present invention or the administration of said compounds to adult subjects. The term "adult" as used herein and in the claims refers to subjects whose globin production has undergone the switch from .gamma.-globin to .beta.-globin; for human subjects, this switch normally takes place around 1-12 weeks after birth.
Perrine U.S. Pat. No. 5,025,029 (issued Jun. 18, 1991) provides a method for ameliorating .beta.-globin disorders in a mammal comprising the step of introducing into the bloodstream of said mammal periodically during its gestation period and/or infancy a compound of the formula: ##STR2## wherein R is --CO.sub.2 R.sub.1, --SO.sub.2 R.sub.1, --SO.sub.3 R.sub.1, or imidazole;
R.sup.' is NH.sub.2, M, H, C.sub.1 -C.sub.4, alkyl or perfluorinated alkyl; PA1 M is a cation; PA1 Z is --CH.sub.3, X, or CX.sub.3 ; PA1 X is H, Cl, Br, I; PA1 Y is H, --NH.sub.2, --NH.sup.+.sub.3, --CX.sub.3 or F; and PA1 R' is H or F. PA1 R.sup.1 is NH.sub.2, M, H, C.sub.1 -C.sub.4, alkyl or perfluorinated alkyl; PA1 M is a cation; PA1 Z is X or CX.sub.3 ; PA1 X is H, Cl, Br, I; PA1 Y is H, --NH.sub.2, --NH.sup.+.sub.3, --CX.sub.3 or F; and PA1 R' is H or F, in an amount sufficient to inhibit development of malarial parasites. This reference does not teach the method of using compounds of the present invention to increase HbF. PA1 (I) XCH.sub.2 --CHX--CHX--C(.dbd.O)--O--Z PA1 (II) CH.sub.3 --CO--CH.sub.2 --C(.dbd.O)--O--Z PA1 (III) CH.sub.3 --CH.sub.2 --CO--C(.dbd.O)--O--Z PA1 (I) XCH.sub.2 --CHX--CHX--C(.dbd.O)--O--Z PA1 (II) CH.sub.3 --CO--CH.sub.2 --C(.dbd.O)--O--Z PA1 (III) CH.sub.3 --CH.sub.2 --CO--C(.dbd.O)--O--Z PA1 X is H, or one of X only may be OH; PA1 Z is --CHR--O--C(.dbd.O)R', --CHR--O--C(.dbd.O)--O--R', or ##STR4## R is H, alkyl, aryl, arylalkyl; and R' is alkyl, aminoalkyl, aralkyl, aryl, alkoxy, aralkoxy and aryloxy, in which aryl by itself, and aryl in aralkyl, aralkoxy and aryloxy are each selected from the group consisting of phenyl, naphthyl, furyl, or thienyl, each of which is unsubstituted or substituted by at least one substituent selected from the group consisting of alkyl, alkoxy, or halogen; PA1 (I) XCH.sub.2 --CHX--CHX--C(.dbd.O)--O--Z PA1 (II) CH.sub.3 --CO--CH.sub.2 --C(.dbd.O)--O--Z PA1 (III) CH.sub.3 --CH.sub.2 --CO--C(.dbd.O)--O--Z PA1 X is H, or one of X only may be OH; PA1 Z is --CHR--O--C(.dbd.O)R', --CHR--O--C(.dbd.O)--O--R', or ##STR5## R is H, alkyl, aryl, arylalkyl; and R' is alkyl, aminoalkyl, aralkyl, aryl, alkoxy, aralkoxy and aryloxy, in which aryl by itself, and aryl in aralkyl, aralkoxy and aryloxy are each selected from the group consisting of phenyl, naphthyl, furyl, or thienyl, each of which is unsubstituted or substituted by at least one substituent selected from the group consisting of alkyl, alkoxy, or halogen; and pharmaceutically acceptable salts and prodrugs thereof.
This patent does not describe the method using compounds of the present invention, or administration of said compounds to adult subjects.
While butyric acid and .alpha.-amino-n-butyric acid treatments have been shown to be quite effective at increasing HbF, these short chain fatty acids have relatively low potency and require prolonged and continuous treatment. These disadvantages make these compounds unattractive as a clinical therapies.
Still other butyrate compounds are presently being studied. Perrine and Faller (Experiencia, 49:133-137, 1993) have examined arginine butyrate. Dover, et. al., (New Eng. J. Med., 327:569-570, 1992) have shown that sodium phenyl acetate and it's prodrug sodium 4-phenylbutyrate are capable of increasing HbF in K562 leukemia cells. Fibach, E., et. al, (Blood, 82: 1-7, 1993) have shown that phenylacetate and 4-phenylbutyrate increase HbF in Erythroid precursor cells. However, all these compounds maintain many of the disadvantages of butyric acid, namely low intrinsic potency, a long induction period, a rapid metabolism and a high clearance. There remains the need for a therapeutic agent capable of enhancing HbF levels, but having a higher potency and low toxicity.
Recently, it has been shown that increasing the level of HbF in a subject is useful for the protection of malaria. HbF inhibits the maturation of the malaria parasites, hemosporidian, in erythrocytes. Perrine U.S. Pat. No. 5,216,004 (issued Jun. 1, 1993) provides a method of preventing malaria in a subject comprising the step of administering to said subject a compound of the formula: ##STR3## wherein R is --CO.sub.2 R.sub.1, --SO.sub.2 R.sub.1, --SO.sub.3 R.sub.1, or imidazole;
Nudelman et al, U.S. Pat. No. 5,200,553 (issued Apr. 6, 1993) described carboxylic acid esters useful to promote antitumor or immune responses selected from the group consisting of compounds having the Formulae (I), (II), or (III):
wherein X is H, or one of X only may be OH; Z is --CHR--O--(O.dbd.)C--R'; R represents a member selected from the group consisting of H and alkyl; and R' represents a member of the group consisting of alkyl, aminoalkyl, aralkyl, aryl, alkoxy, aralkoxy and aryloxy, in which aryl by itself, and aryl in aralkyl, aralkoxy and aryloxy are each selected from the group consisting of sub-groups (a) and (b), wherein (a) is unsubstituted phenyl, naphthyl, furyl, or thienyl and (b) is phenyl, naphthyl, furyl, or thienyl, each of which is substituted by at least one substituent selected from the group consisting of alkyl, alkoxy, or halogen, provided that in (I) when X is H and R' is propyl, then R is alkyl which contains at least three carbon atoms.
None of the above references teach or suggest the method of using carboxylic acid compounds of Formulae (I-III) to increase the level of HbF in vitro, or in vivo in subjects and particularly in adult subjects. Accordingly, an object of the present invention is the method of increasing the level of HbF comprising administering one or more compounds of Formulae (I-III). It is a further object of this invention to provide a method for increasing HbF levels in adults in need of such treatment without toxicity and with greater potency. Still a further object of this invention is to provide a method of increasing HbF in vitro for diagnostic purposes comprising utilizing a compound of the Formulae (I-III). The methods of the present invention are particularly useful for preventing or ameliorating the clinical effects of various disorders by increasing the level of HbF in subjects afflicted with such anomalies. Such disorders include but are not limited to globin disorders (such as sickle cell anemia and .beta.-thalassemia) and malaria.